T lymphocytes bearing alpha-beta receptors respond to complexes of peptide antigen and class I or class II MHC molecules. The way in which protein antigens are transformed into peptides suitable for such binding, the events involved in facilitating such peptide-MHC association, and the pathways followed by MHC molecules both before and after peptide association are critical to our understanding of T cell immunity. We previously proposed two fundamentally distinct pathways for peptide acquisition by class I and class II MHC molecules. Substantial functional data support this model, although important exceptions exist. To examine the biochemical and cell biological basis for this distinction, and the allele-independent molecular events involved in peptide-MHC molecule association, we have examined normal and mutant cells lines varying in their capacity to generate effective peptide-MHC complexes for the rate of class I and class II molecule assembly and transport, in the presence and absence of known MHC ligand peptides, and under varying growth conditions. These studies have revealed that peptide-class I MHC interaction involves what can be considered an "allosteric" interaction among peptide, MHC class I heavy chain, and beta2-microglobulin (see also the report of the Molecular Biology Section, LI, NIAID), that results in a stabilization of the ternary complex and resistance to thermal denaturation. This process appears to be localized to the endoplasmic reticulum, and accounts for the predilection of class I molecules for peptides generated from intracellular proteins. Class II molecules do not share this requirement for peptide for thermally stable chain association, but rather are regulated in their folding and peptide binding capacity by additional molecules, including the invariant chain (see Z01 AI 00349-07 LI). However, in transfected cells, it has not been possible to show a functional role for li with respect to the efficiency of antigen processing. These data suggest a more complex role for li and specialized physiology in class II-expressing, antigen-presenting cells than previously postulated.